1. Field of the Invention
The present invention generally relates to anti-hepatitis B virus surface protein antibodies, immunoassay kit comprising the same and method of detecting hepatitis B virus surface proteins, in particular to a hepatitis B virus surface protein-specific antibody, a large hepatitis B virus surface protein-specific antibody, a wild type large hepatitis B virus surface protein-specific antibody, an immunoassay kit comprising the antibodies, and a method of detecting pre-S2 deletion mutant large hepatitis B virus surface protein.
2. Description of the Related Art
Chronic hepatitis B virus infection is a major cause of hepatocellular carcinoma (HCC) worldwide and its most important cause in Asia. Hepatitis B virus (HBV)-related HCC often occurs at the age of 40 or older, suggesting that HBV may persist in carriers for decades before HCC actually develops. It is important for long-term monitoring high-risk markers in chronic HBV carriers to identify the ones that need frequent follow-up or uptake prophylactic therapies. In addition, development of high-risk recurrence markers in HCC patients receiving hepatectomy surgery is also important for clinicians to identify the ones with relatively worse prognosis, who need to uptake aggressive treatments. Though up to now the methods (e.g. ultrasound) and tumor markers (e.g. alpha-fetal protein) to diagnose HCC have been established, the high-risk markers for HCC incidence and recurrence have not been fully identified, given that HCC tumorigenesis is a complex process regulated by various crosstalks between host and viral factors.
The pre-S2 deletion mutant large hepatitis B virus surface protein (LHBS), isolated from the type 2 ground glass hepatocyte (GGH), is highly associated with HCC (1). GGH is prevalent in livers with chronic HBV infection and HBV-induced HCC tumors. Type I GGH are usually scattered singly in the hepatic lobules with the expression of dense homogeneous or “inclusion-like” pattern of HBS. This type of GGH usually appears starting at the early carrier stage or in patients with active diseases, frequently co-expresses with a nuclear or cytoplasmic core antigen (2,3). Previous studies found that type I GGH harbors the LHBS partially deleted of the pre-S1 region and accumulates in ER lumen (4). On the other hand, type II GGH expresses a unique pattern of HBS at the cell margin or periphery (For GGH morphologies, see FIG. 1). Most interestingly and importantly, this type of GGH consistently clusters in nodules and usually appear at the advanced or low replicative stages of virus replication, and are frequently associated with cirrhosis or HCC, which suggests that type II GGH may represent clonally-proliferated, adenomatous, or preneoplatic lesions of HCC (2,3). By dissecting the cirrhotic nodules containing type II GGH, the HBV genomes were clonally proliferated and integrated, supporting the concept of a clonal or adenomatous lesion of type II GGH. This type of GGH displays a unique pattern of “marginal” type surface antigens and harbors the LHBS partially deleted of the pre-S2 region, designated pre-S2 mutant LHBS (3). The pre-S2 mutant LHBS is highly expressed in most of the HBV-induced cirrhotic nodules through the stages of the pre-neoplastic lesions, early HCC, and large HCC tumors, which implies the important role of pre-S2 mutant LHBS in the tumor progression of HCC. The pre-S2 deletion region clusters at the N-terminus of the pre-S2 region, predominantly in the range of 40 to 60 nucleotides long, although some cases contain deletions of as short as 10 nucleotides and still present type II GGH morphology (3). Some of the pre-S2 mutant LHBS also harbors a point mutation at the pre-S2 ATG start codon, causing no synthesis of the middle S antigen (FIG. 2). The region commonly deleted in the pre-S2 mutant LHBS contains a CD8 T-cell epitope, which proposes a hypothesis that this type of mutant LHBS escapes from immune surveillance then becomes a selective clone in long-term persistence of HBV infection.
In the past 10 years or so, the inventors of the present invention focused on studying the pathological effects caused by the pre-S mutant LHBS, especially the pre-S2 type, since this type of LHBS is particularly highly associated with HCC. It was found that the both of the pre-S1 and -S2 mutant LHBS distribute in endoplasmic reticulum (ER) of hepatocytes and induce ER stress (4). However, the pre-S2 mutant LHBS specifically induces ER stress-induced growth proliferation mediated through the NFκB pathway (5). The pre-S2 mutant LHBS also induces significantly higher level of ER stress-dependent oxidative stress and oxidative DNA damages than the wild-type or pre-S1 mutant LHBS do, which leads to genomic instability and consequently hepatocellular carcinogenesis (6). In addition, the pre-S2 mutant LHBS specifically induces cyclin A over-expression (7) and directly interacts with Jun activation domain-binding protein 1 (JAB1) to cause inactivation of the tumor suppressor retinoblastoma and cell cycle progression (8). Together these findings have offered clear lines of evidence of that the pre-S2 mutant LHBS likely plays an important role in HBV-induced HCC.
The pre-S2 mutant LHBS is relatively low at acute phase of HBV infection but greatly increased in the long-term period of HBV infection. In HCC, it is higher than 60%, which indicates that it is highly prevalent in late phase of chronic HBV infection and significantly associated with advanced liver diseases such as cirrhosis and HCC (9). To detect the pre-S deletion in LHBS, we previously developed a Pre-S Gene Chip, which detects the pre-S deletion based on DNA hybridization of the HBS genes in patients to the DNA probes on the chip. It was found that the WT and pre-S mutant LHBS often co-exist in one individual HBV carrier, which makes the detection of the pre-S mutant difficult. Moreover, it requires labor-intensive process for cloning and isolating each individual gene product before the chip analysis.
Furthermore, the pre-S2 deletion mutants have also been shown to increase after anti-viral therapies using nucleoside analogs to chronic HBV carriers, suggesting that the carriers presenting the pre-S2 mutant large HBV surface (LHBS) protein are the ones in high risk to HCC. See Zhang et al. BMC Microbiology, 12: 307-316 (2012). Thus, there is a need to develop a rapid and accurate method for detecting deletions in the HBV Pre-S region, thereby assessing a HBV carrier's risk of developing cirrhosis/HCC.